Slope spectroscopy standards

ABSTRACT

The present invention relates generally to a slope spectroscopy standards and methods of making slope spectroscopy standards, specifically standards and methods of developing standards specifically for variable pathlength (slope) measurements.

RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 62/762,891 which wasfiled on May 24, 2018.

FIELD OF THE INVENTION

The present invention relates generally to a slope spectroscopystandards and methods of making slope spectroscopy standards,specifically standards and methods of developing standards specificallyfor variable path length (slope) measurements.

BACKGROUND OF THE INVENTION

Spectroscopic analysis is a broad field in which the composition andproperties of a material in any phase, gas, liquid, solid, aredetermined from the electromagnetic spectra arising from the interaction(eg. absorption, luminescence, or emission) with energy. One aspect ofspectrochemical analysis, known as spectroscopy, involves interaction ofradiant energy with the material of interest. The particular methodsused to study such matter-radiation interactions define many sub-fieldsof spectroscopy. One field in particular is known as absorptionspectroscopy, in which the optical absorption spectra of liquidsubstances are measured. The absorption spectrum is the distribution oflight attenuation (due to absorbance) as a function of light wavelength.In a simple spectrophotometer the sample substance which is to bestudied is placed in a transparent container, also known as a cuvette orsample cell. Electromagnetic radiation (light) of a known wavelength, λ,(ie. ultraviolet, infrared, visible, etc.) and intensity I is incidenton one side of the cuvette. A detector, which measures the intensity ofthe exiting light, I is placed on the opposite side of the cuvette. Thelength that the light propagates through the sample is the distance d.Most standard UV/visible spectrophotometers utilize standard cuvetteswhich have 1 cm path lengths and normally hold 50 to 2000 μL of sample.For a sample consisting of a single homogeneous substance with aconcentration c, the light transmitted through the sample will follow arelationship know as Beer's Law: A=εcl where A is the absorbance (alsoknown as the optical density (OD) of the sample at wavelength λ whereOD=the—log of the ratio of transmitted light to the incident light), εis the absorptivity or extinction coefficient (normally at constant at agiven wavelength), c is the concentration of the sample and l is thepath length of light through the sample.

Spectroscopic measurements of solutions are widely used in variousfields. Often the compound of interest in solution is highlyconcentrated. For example, certain biological samples, such as proteins,DNA or RNA are often isolated in concentrations that fall outside thelinear range of the spectrophotometer when absorbance is measured.Therefore, dilution of the sample is often required to measure anabsorbance value that falls within the linear range of the instrument.Frequently multiple dilutions of the sample are required which leads toboth dilution errors and the removal of the sample diluted for anydownstream application. It is, therefore, desirable to take existingsamples with no knowledge of the possible concentration and measure theabsorption of these samples without dilution.

Multiple sample cuvettes may solve the problem of repetitive sampling,however, this approach still requires the preparation of multiple samplecuvettes and removes some sample from further use. Furthermore, in mostspectrophotometers the path length, l, is fixed.

Another approach to the dilution problem is to reduce the path length inmaking the absorbance measurement. By reducing the measurement pathlength, the sample volume can be reduced. Reduction of the path lengthalso decreases the measured absorption proportionally to the path lengthdecrease. For example, a reduction of path length from the standard 1 cmto a path length of 0.2 mm provides a virtual fifty-fold dilution.Therefore, the absorbance of more highly concentrated samples can bemeasure within the linear range of the instrument if the path length ofthe light travelling through the sample is decreased. There are severalcompanies that manufacture cuvettes that while maintaining the 1 cm²dimension of standard cuvettes decrease the path length through thesample by decreasing the interior volume. By decreasing the interiorvolume less sample is required and a more concentrated sample can bemeasured within the linear range of most standard spectrophotometers.While these low volume cuvettes enable the measurement of moreconcentrated samples the path length within these cuvettes is stillfixed. If the sample concentration falls outside the linear range of thespectrophotometer the sample still may need to be diluted or anothercuvette with an even smaller path length may be required before anaccurate absorbance reading can be made.

While some of these instruments provide the capability of varying thepath length for measurement of highly concentrated low volume samplesthe applications described therein relate primarily to single pathlength and single wavelength measurements. Several of the instrumentsprovide a limited number of path lengths and all are limited to pathlength larger than 0.2 mm. Furthermore, the devices and methods of theprior art do not provide for expanding the dynamic range of thespectrophotometer so that it is not necessary to adjust theconcentration of the sample to fall within the linear range ofabsorbance detection of the instrument. To the extent that the prior artteaches shorter path lengths to determine the concentration of veryconcentrated samples or low volume samples the focus of these devices isto take a single absorbance reading at a single path length. As such theprior art references require that the path length be known with greataccuracy so that an accurate concentration measurement can be made.

Historically, various liquid standard solutions have been used asaccurate absorbance standards, the most common of these being potassiumdichromate in various media. For example, one standard that has beenused is 57.0 to 63.0 mg of potassium dichromate in 0.005M sulphuric aciddilute to 1000 ml to test absorbance at 235, 257, 313 and 350 nmwavelengths. A tenfold more concentrated solution of potassiumdichromate has been used to provide an additional test point at 430 nm.The 1%/1 cm value is recorded and checked against the target range.Other solutions such as nictonic acid in 0.1M hydrochloric acid havebeen used to assess photometric accuracy in the far UV region. In recentyears these solutions have been replaced by the use of neutral densityglass filters which may be calibrated and traced to internationallyrecognized standards such as the National Institute of Standards andTechnology (NIST).

A slope spectrometer, such as the SoloVPE™ and the Slope Spectroscopy™provides devices and methods that provide a variable path lengthspectrophotometer which dynamically adapts parameters in response toreal time measurements via software control to expand the dynamic rangeof a conventionally spectrophotometer such that samples of almost anyconcentration can be measured without dilution or concentration of theoriginal sample. Using existing UV Vis Standards for the testing andqualification of slope spectroscopy systems is not an ideal situation asthese standards were designed for absorbance not slope measurements. Ifthe conventional standard is designed to be measured at a 1 mmpathlength the slope value should effectively be the certifiedabsorbance value, but these are fundamentally different measurements, ondifferent pieces of equipment. As such the uncertainties reported on thecertificates for exiting UV Vis Absorbance Standards are not trulytransferable to the slope based technology. Thus, the present inventionrelates to standards and methods of developing standards specificallyfor variable pathlength (slope) measurements.

SUMMARY OF THE INVENTION

The present invention relates to methods making a slope spectroscopystandard for a compound by selecting a compound of interest anddetermining the compound slope at a predetermined concentration and thendetermining a concentration of a second compound which corresponds tothe compound of interest slope by using variable path lengthspectroscopy and then making a standard from the second with the slopethat corresponds to the compound of interest at that particularconcentration.

The present invention also relates to methods of making slopespectroscopy standards for a combination of compounds that have aparticular ratio of one compound and another compound in a combinationof compounds by determining the ratio of the two compounds in thecombination of the compounds and determining a concentration of firstsubstance which absorbs light at a wavelength corresponding to the firstcompound corresponding to the first compound by using variable pathlength spectroscopy and determining a concentration of a secondsubstance which absorbs light at a wavelength corresponding to thesecond compound corresponding to the second compound slope by usingvariable path length spectroscopy and making a first substance/secondsubstance standard by combining the first substance at a concentrationwith the slope corresponding to the first compound with the secondsubstance at a concentration with the slope corresponding to the secondcompound at a ratio corresponding to the first compound/secondcombination.

The present invention relates to methods of making a slope spectroscopystandard for an antibody/drug combination of a particular ratio of drugto antibody by determining the antibody to drug ratio of theantibody/drug combination and determining a concentration of caffeinecorresponding to the antibody by using variable path length spectroscopyand determining a concentration of a compound which absorbs light at awavelength corresponding to the drug corresponding to the drug slope byusing variable path length spectroscopy and making a caffeine/compoundstandard by combining the caffeine at a concentration with the slopecorresponding to the antibody with the compound at a concentration withthe slope corresponding to the drug at a ratio corresponding to theantibody/drug combination. The compound can be a colorant which absorbslight at a wavelength corresponding to the drug or the compound can be amicrobead which absorbs light at a wavelength corresponding to the drug.

The present invention also relates to methods for determining the stateof fitness of a spectrophotometer by testing the spectrophotometer usinga caffeine standard and measuring the slope using variable path lengthspectroscopy and then making adjustments to the spectrophotometer andre-testing the spectrophotometer using a caffeine standard and measuringthe slope using variable path length spectroscopy and comparing theresults of the test with the re-test to determine the state of fitnessof the spectrophotometer.

FIGURES

FIG. 1 is an absorbance spectrum for caffeine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to standards that are used in devices andmethods for determining the spectrophotometric characteristics of asolution by the use of a variable path length. For example, indetermining the concentration of a compound in solution the absorbanceof the solution can be determined at various path lengths. The values ofthe absorbance at various path lengths can then be used to calculate theconcentration of the compound in the solution which are particularlyuseful for determining the concentration of highly concentrated sampleswithout resorting to single or multiple dilutions of the samples. Inessence these devices and methods expand the dynamic range of a standardspectrophotometer by permitting a wide range of path lengths formeasuring the absorbance values of a solution. This broad dynamic rangeenables users to determine the concentrations of their samples withoutaltering (diluting or concentrating) the samples. Variable path lengthdevices may comprise a probe tip, sample vessel, motor, delivery opticalfiber, detector, unidirectional sliding mechanism and appropriatesoftware for path length control and measurement parameters.

The variable path length device may be used to measure the concentrationof a sample by placing the solution sample to be tested in a samplevessel such as but not limited to a cuvette and determining theabsorbance at a predetermined wavelength corresponding to the samplesolution at a predetermined path length and repeating the absorbancemeasurement at different path lengths. Once a number of absorbancevalues are obtained at different path lengths a regression line can begenerated from the absorbance and path length such that a slope of theregression line is obtained and the concentration of the sample may beobtained by dividing the slope of the regression line by the extinctioncoefficient of the sample.

Previously existing UV Vis Standards was used for the testing andqualification of these variable path length systems that utilized slopespectroscopy. However, these standards were not ideal as the UV VisStandards were designed for absorbance measurements and not slopemeasurements. If a UV Vis standard is designed to be measured at a 1 mmpath length the slope value should effectively be the certifiedabsorbance value, but absorbance measurements and slope measurements arefundamentally different measurements that are determined using differentpieces of equipment. As such the uncertainties reported on thecertificates for US Vis standards are not truly transferable to theslope based technology. Therefore, it became necessary to developstandards specifically designed for variable path length (slope)measurements.

The standards of the present invention may be any compound that whenmeasured at a given wavelength, create a change in absorbance atdifferent path lengths and for which a slope can be generated. Oneexample is caffeine which may be used as a standard for samples thatabsorb in the range of 280 nm, such as proteins. Other examples ofcompounds that can be as standards include but are not limited to NaClwhich may be used to calibrate standards for salt water; KHP which maybe used with water to check the pH of solutions; KCl which may be usedas a salt for conductivity measurements; and KCr2O4 which is a standardfor UV/Vis instruments

In selecting an appropriate standard for a given compound it isdesirable that the standard is sufficiently soluble or at least in thecase of suspensions homogeneous in the given solution such that ameasurable slope may be obtained. In the standards of the presentinvention the slope measured should be at least 0.01 abs/mm or at least0.1 abs/mm or at least 0.5 abs/mm or at least 1.0 abs/mm or at least 5.0abs/mm or at least 10.0 abs/mm or at least 25.0 abs/mm or at least 50.0abs/mm or at least 100 abs/mm or at least 150 abs/mm or at least 200abs/mm or at least 250 abs/mm or at least 300 abs/mm or at least 350abs/mm or at least 400 abs/mm. In the standards of the present inventionthe slope measured should be from about 0.01 abs/mm to about 400 abs/mm,or from about 0.01 abs/mm to about 300 abs/mm or from about 0.01 abs/mmto about 200 abs/mm or from about 0.01 abs/mm to about 100 abs/mm orfrom about 0.01 abs/mm to about 50 abs/mm or from about 0.01 abs/mm toabout 25.0 abs/mm or from about 0.01 abs/mm to about 10 abs/mm or fromabout 0.01 abs/mm to about 5 abs/mm or from about 0.1 abs/mm to about400 abs/mm, or from about 0.1 abs/mm to about 300 abs/mm or from about0.1 abs/mm to about 200 abs/mm or from about 0.1 abs/mm to about 100abs/mm or from about 0.1 abs/mm to about 50 abs/mm or from about 0.1abs/mm to about 25.0 abs/mm or from about 0.1 abs/mm to about 10 abs/mmor from about 0.1 abs/mm to about 5 abs/mm or from about 1 abs/mm toabout 400 abs/mm, or from about 1 abs/mm to about 300 abs/mm or fromabout 1 abs/mm to about 200 abs/mm or from about 1 abs/mm to about 100abs/mm or from about 1 abs/mm to about 50 abs/mm or from about 1 abs/mmto about 25.0 abs/mm or from about 1 abs/mm to about 10 abs/mm or fromabout 1 abs/mm to about 5 abs/mm or from about 5 abs/mm to about 400abs/mm, or from about 5 abs/mm to about 300 abs/mm or from about 5abs/mm to about 200 abs/mm or from about 5 abs/mm to about 100 abs/mm orfrom about 5 abs/mm to about 50 abs/mm or from about 5 abs/mm to about25.0 abs/mm or from about 5 abs/mm to about 10 abs/mm or from about 10abs/mm to about 400 abs/mm, or from about 10 abs/mm to about 300 abs/mmor from about 10 abs/mm to about 200 abs/mm or from about 10 abs/mm toabout 100 abs/mm or from about 10 abs/mm to about 50 abs/mm or fromabout 10 abs/mm to about 25.0 abs/mm. In preferred embodiments of thestandards of the present invention the standard does not substantiallydegrade over time such that the slope measurement is constant over time.In some embodiments of the standards of the present invention, thestandard in a “sealed” condition may have a shelf life of 3 months ormore, or 6 months or more or 1 year or more or 2 years or more or 5years or more. The working life of the standard should also have auseful life while being used during a test, such that there is littledegradation over a span of ten minutes or more. The standard solutionshould be stable over the time it takes to use the solution. Since thestandard may be water based it may start to evaporate and thus changeover time if left exposed to the environment.

One example of a slope spectroscopy standard of the present invention iscaffeine in water as it provides a user-friendly material with excellentstability, great solubility and customization across a range of slopevalues. In one embodiment of the standards of the present invention astock solution of roughly 10,000 ppm or 10 mg/ml concentration of purecaffeine in water is made. From that stock the caffeine solution isdiluted to a predetermined target range that corresponds either to apredetermined standard slope or a custom standard slope that is createdbased on a particular compound or on a multi-component product such asan antibody drug conjugate. The standards of the present inventionprovide a certified slope value with an uncertainty that is NISTtraceable. This is done in accordance with ISO 9001:2008 QualityManagement System and in compliance with the ISO 17025:2005 and ISOGuide 24:2009 standards. The measurement and certification is based upona version of variable path length technology that has closed loopcontrol of the path length and high resolution.

In pharmaceutical and biological processes the concentration ofsubstances including that active pharmaceutical compound or biologicalsubstance within fluid samples may be measured intermittently orcontinuously during and after processing or purification of the compoundof interest. It is of great interest to know with great precision andaccuracy the concentration of any given substance at any time in theprocess. It is especially important to be able to quantify the finalproduct of a process especially when that product is being sold to thepublic for treatment of diseases. Having a standard that ensures thatthe concentration of the solution, as measured by the spectrophotometricinstrument, is an essential part of good quality management.

With any standard there is a certain level of uncertainty. The source ofthat uncertainty may be related to a variety of factors including theinstrument used, homogeneity of the sample, the variation of the samplevessel and other factors. Obviously, the better the instrument formeasuring the sample and the more homogenous the sample, the lower theuncertainty of the measurement. Often, when reporting uncertainty thevalues, are usually reported in the context of the instrument whichmeans the assumption is that the uncertainty value is the same for allinstruments. This approach is simplistic and inaccurate. For examplethere can be a standard with a reported uncertainty value of <1%uncertainty but on a high-end instrument. This value will not be thesame on a lesser instrument. Furthermore, the uncertainty likely varieswith varying slope numbers. A lower slope value will have larger stepchanges and probably less error, while a larger slope value will havesmall step changes and therefore a larger error. The standards of thepresent invention may be used at a particular wavelength at which thestandard absorbs light or can be used at a wavelength near where thestandard absorbs light. The uncertainty increases as the wavelength usedis further from the optimum absorbance wavelength.

Measured Slope vs. Certified Slope Value: The Certified Slope Value isdetermined by a statistically based uncertainty determination method andtakes into account a number of variables (controlled and uncontrolled).The value is typically expressed with a 95% confidence. When using theSlope Standard a comparison is made between the measured slope and thecertified slope value. If the measured value(s) do not fall within arange that is set by combining the uncertainty of the standard and theslope uncertainty of the instrument being evaluated, and all othersources of potential variation (e.g. cleanliness, method, handling etc.)have been ruled out, the system is likely not performing as intended.

Standard Deviation of the Measured Slope Values (1-→n): Standarddeviation or other statistical techniques (e.g. RMS, MSD, analysis ofresiduals, etc.) may be used to evaluate the repeatability of multiplemeasurements and the spread of the data. Using a known sample with acertified slope value serves as an excellent benchmark for assessing therepeatability performance of the system which has a known value.Depending on the spectroscopic device being evaluated that value couldrange from ±1 to ±5. The benefit of the standard is having a sample ofknown performance and stability so performance can be determined withhigher confidence. The actual threshold value used that may be used todiagnose an issue can vary depending on the device potentially thenominal slope value of the standard itself. These statistical methodsare a way of determining the ariability between slope measurements.

Linearity Results (Strength of Correlation) R²: While the standarddeviation of the measure slope values is a metric for determining thevariability between slope measurements, linearity results look atperformance variability within a single measurements based upon multipleabsorbance values at different path lengths. Using a known standard witha known method should provide a highly predictable result based upon thecapability of the instrument being assessed. A poor correlation,represented by low R² values, may indicate an issue with the system.Typically methods are developed to deliver a minimum R² value of 0.999with a minimum number of data points. The exact method used with eachstandard may be based upon method development and validation work forthe various slope values being produced. Linearity deviations canindicate multiple issues that may involve photometric issues with thesystem or potentially the motion control of the system. Sometimecleanliness of the system is the issue. With linearity the R2 value anddata point count will need to meet some minimum level of expectations.If non-linear points appear or need to be excluded, which is atypicalfor the method, there is likely a problem.

Absorbance Range (Min/Max Values): While the photometric range of thespectrophotometer engine is set, issues of optical coupling, lamp age,cleanliness and alignment can all influence the amount of lighttransmitted through the system. While this value may be unique for eachsystem, it should generally fall within a certain predicatable range.The absorbance result for a given standard is could indicate whetherthere is more or less light than would be expected for a given slopevalue (concentration). It is more probable that less light is traversingthe path length which will indicate a problem, however, the possibilityof too much light could reveal a stray light issue.

In the most basic example of the standards of the present invention, apredetermined concentration of a standard can be analyzed by variablepath length spectroscopy to determine the slope of that concentration asdescribed above. In one embodiment of the methods of the presentinvention serial dilutions of the compound chosen for the standard canbe used to create a standard curve of slopes “book ending” the range ofslopes. This step may be accomplished using a robotic system set up fordiluting. From this data a concentration can be chosen for a desiredslope. The tolerance for the desired slope can be determined. It isimportant that whether the predetermined slope is 5 and the standard is4.9, that the standard 4.9 is NIST traceable.

The uncertainty for any given standard should be less than 10% or lessthan 9% or less than 8% or less than 7% or less than 6% or less than 5%or less than 4% or less than 3% or less than 2% or less than 1.5% orless than 1.4% or less than 1.3% or less than 1.2% or less than 1.1% orless than 1.0% or less than 0.9% or less than 0.8% or less than 0.7% orless than 0.6% or less than 0.5% or less than 0.4% or less than 0.3% orless than 0.2% or less than 0.1%. The uncertainty for any given standardshould be between about 0.05% to about 10% or from about 0.05% to about9% or from about 0.05% to about 8% or from about 0.05% to about 7% orfrom about 0.05% to about 6% or from about 0.05% to about 5% or fromabout 0.05% to about 4% or from about 0.05% to about 3% or from about0.05% to about 2% or from about 0.05% to about 1.5% or from about 0.05%to about 1.4% or from about 0.05% to about 1.3% or from about 0.05% toabout 1.2% or from about 0.05% to about 1.1% or from about 0.05% toabout 1.0% or from about 0.05% to about 0.9% or from about 0.05% toabout 0.8% or from about 0.05% to about 0.7% or from about 0.05% toabout 0.6% or from about 0.05% to about 0.5% or from about 0.05% toabout 0.4% or from about 0.05% to about 0.3% or from about 0.05% toabout 0.2% or from about 0.05% to about 0.1% or from about 0.1% to about10% or from about 0.1% to about 9% or from about 0.1% to about 8% orfrom about 0.1% to about 7% or from about 0.1% to about 6% or from about0.1% to about 5% or from about 0.1% to about 4% or from about 0.1% toabout 3% or from about 0.1% to about 2% or from about 0.1% to about 1.5%or from about 0.1% to about 1.4% or from about 0.1% to about 1.3% orfrom about 0.1% to about 1.2% or from about 0.1% to about 1.1% or fromabout 0.1% to about 1.0% or from about 0.1% to about 0.9% or from about0.1% to about 0.8% or from about 0.1% to about 0.7% or from about 0.1%to about 0.6% or from about 0.1% to about 0.5% or from about 0.1% toabout 0.4% or from about 0.1% to about 0.3% or from about 0.1% to about0.2% or from about 0.5% to about 10% or from about 0.5% to about 9% orfrom about 0.5% to about 8% or from about 0.5% to about 7% or from about0.5% to about 6% or from about 0.5% to about 5% or from about 0.5% toabout 4% or from about 0.5% to about 3% or from about 0.5% to about 2%or from about 0.5% to about 1.5% or from about 0.5% to about 1% or fromabout 1% to about 10% or from about 1% to about 9% or from about 1% toabout 8% or from about 1% to about 7% or from about 1% to about 6% orfrom about 1% to about 5% or from about 1% to about 4% or from about 1%to about 3% or from about 1% to about 2% or from about 2% to about 10%or from about 2% to about 9% or from about 2% to about 8% or from about2% to about 7% or from about 2% to about 6% or from about 2% to about 5%or from about 2% to about 4% or from about 2% to about 3% or from about3% to about 10% or from about 3% to about 9% or from about 3% to about8% or from about 3% to about 7% or from about 3% to about 6% or fromabout 3% to about 4%.

In other embodiments of the present invention a standard for the slopefor a particular compound of interest at a particular concentration maybe developed by determining the concentration of a standard whichprovides the identical slope value to that of particular compound ofinterest at the particular concentration. For example, it may bedesirable to have a standard which mimics the slope of a commercial drugcompound at the concentration of the drug in the final product. Havingthe standard for that particular drug at that particular concentrationcan provide a release test for that drug as long as the drug is withinthe error range of the desired concentration.

In other embodiments of the present invention a standard for the slopesof a particular combination of compounds such as antibody drugconjugates is desirable. Antibody-drug conjugates or ADCs are highlypotent biopharmaceutical drugs designed as targeted drug therapy for thetreatment of people with cancer. ADCs are complex molecules composed ofan antibody linked, via a stable, chemical linker with labile bonds, toa biological active cytotoxic compound. The manufacture of ADCs providesthe challenge of conjugating the cytotoxic drug component to theantibody via a chemical linker in a reproducible fashion. It isdesirable to characterize the drug to antibody ratio, the amount ofbound drug versus unbound drug, the amount of unbound linker and todetermine the stability of the ADC both in vitro and in vivo. For thesecompounds a primary goal is to ensure that the ratio of drug to antibodyfalls within a targeted range. To mimic the antibody-drug combinationthe standards of the present invention may use a combination of amaterials such as caffeine and a compound that absorbs light at awavelength similar to a drug. Such a compound may be but is not limitedto a stable colorant, micro-beads or off peak absorber of another typeto allow variable path length users to have a certified drug:antibodyratio that can be measured for qualification and system suitability.

Within this disclosure, any indication that a feature is optional isintended provide adequate support (e.g., under 35 U.S.C. 112 or Art. 83and 84 of EPC) for claims that include closed or exclusive or negativelanguage with reference to the optional feature. Exclusive languagespecifically excludes the particular recited feature from including anyadditional subject matter. For example, if it is indicated that A can bedrug X, such language is intended to provide support for a claim thatexplicitly specifies that A consists of X alone, or that A does notinclude any other drugs besides X. “Negative” language explicitlyexcludes the optional feature itself from the scope of the claims. Forexample, if it is indicated that element A can include X, such languageis intended to provide support for a claim that explicitly specifiesthat A does not include X. Non-limiting examples of exclusive ornegative terms include “only,” “solely,” “consisting of,” “consistingessentially of,” “alone,” “without”, “in the absence of (e.g., otheritems of the same type, structure and/or function)” “excluding,” “notincluding”, “not”, “cannot,” or any combination and/or variation of suchlanguage.

Similarly, referents such as “a,” “an,” “said,” or “the,” are intendedto support both single and/or plural occurrences unless the contextindicates otherwise. For example “a dog” is intended to include supportfor one dog, no more than one dog, at least one dog, a plurality ofdogs, etc. Non-limiting examples of qualifying terms that indicatesingularity include “a single”, “one,” “alone”, “only one,” “not morethan one”, etc. Non-limiting examples of qualifying terms that indicate(potential or actual) plurality include “at least one,” “one or more,”“more than one,” “two or more,” “a multiplicity,” “a plurality,” “anycombination of,” “any permutation of,” “any one or more of,” etc. Claimsor descriptions that include “or” between one or more members of a groupare considered satisfied if one, more than one, or all of the groupmembers are present in, employed in, or otherwise relevant to a givenproduct or process unless indicated to the contrary or otherwise evidentfrom the context.

Where ranges are given herein, the endpoints are included. Furthermore,it is to be understood that unless otherwise indicated or otherwiseevident from the context and understanding of one of ordinary skill inthe art, values that are expressed as ranges can assume any specificvalue or subrange within the stated ranges in different embodiments ofthe invention, to the tenth of the unit of the lower limit of the range,unless the context clearly dictates otherwise.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. The citation of any publication is for its disclosure priorto the filing date and should not be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that the various changes in form and detailsmay be made therein without departing from the scope of the inventionencompassed by the appended claims.

A better understanding of the present invention and of its manyadvantages will be had from the following examples, given by way ofillustration.

We claim:
 1. A method of making a slope spectroscopy standard for anantibody/drug combination of a particular ratio of drug to antibodycomprising: determining the antibody to drug ratio of the antibody/drugcombination; determining a concentration of first compound which absorbslight at a wavelength corresponding to the antibody by using variablepath length spectroscopy; determining a concentration of a secondcompound which absorbs light at a wavelength corresponding to the drugcorresponding to the drug slope by using variable path lengthspectroscopy; making a first compound/second compound standard bycombining the first compound at a concentration with the slopecorresponding to the antibody with the second compound at aconcentration with the slope corresponding to the drug at a ratiocorresponding to the antibody/drug combination.
 2. The method of claim1, wherein the first compound is caffeine.
 3. The method of claim 1wherein the second compound is a colorant which absorbs light at awavelength corresponding to the drug.
 4. The method of claim 1 whereinthe compound is a second microbead which absorbs light at a wavelengthcorresponding to the drug.